Team:ULaval/Human Practices/ExpertInsight
We presented our project to teachers of our faculty, and they came up with advice to help us improve our design.
Dr. Caroline Duchaine
An international expert in bio-aerosols, told us that it would be more relevant to target microorganisms which should not be found in an environment at all, therefore with a tolerance threshold of 0 PPM, rather than targeting microorganisms which are tolerated underneath a certain threshold. This was a very good word of advice, as targeting tolerated microorganisms would have required an efficient quantification method, which was not compatible with the technology we chose. She also suggested to couple our machine with always characterized liquid impactors such as a Vortex, instead of a classical impactor. We were also told to keep in mind the size of targeted particles to make sure that they would reach our detector. She disapproved of the idea of putting a filter at the entry of our system, which ended up with us adding the sonication part to break up the particles so they would be compatible with the microfluidics device, while also removing a potential cause for false-negatives. The presence of a filter could have retained larger particles such as clumps of microorganisms, which would have been filtered out and not been detected. She also thought that a proof of concept using a eukaryotic virus would be interesting and that using antibodies to target capsid proteins of viruses rather than their genome could make detection easier. However, the use of antibodies in such a device would have made it much costlier and making antibodies can be very difficult and expensive. She continued by saying that it could be a good idea to contact cruising companies who could be interested in our project and to think about the possibility of using a photomultiplier as a detector for our system. Finally, she shared with Dr. Michel Guertin the idea that we should study DNA viruses to facilitate lab work. Our initial focus was a model of the Norovirus, an RNA-based virus, and working with viral gRNA can be very tricky. We, therefore, chose to focus our efforts on DNA and mRNA, which a generally easier to work with.
Dr. Michel Guertin
Expressed doubts about the need for the in vivo steps in our project. In the end, we decided to heed this comment, and did all characterization tests in vitro, as this was more representative of how our tool would end up working. He advised us to use commercial cell-free expression systems, which we did. At first, we wanted to reduce costs to a maximum by making our own cell-free system, but this would have added a step of complexity to our design. In the end, we managed to get a very generous sponsorship from ArborBiosciences, which allowed us to work with an extremely efficient cell-free expression system. Finally, he advised us that we should ask the people in Engineering for help with the construction of our microfluidic chip. We contacted this department, and we worked with a student in the Industrial Engineering Department and an expert in microfluidics from the Chemistry Department, which helped us tremendously to make and optimize our circuit.
Dr. Manon Couture
Thought that we should consider the propagation of the wave from the sonication step on the rest of the chip and to look for whether this had been characterized. We decided to move the sonication step to the outside of the microfluidics device, as it could have affected the integrity of the chips and therefore the sample. This made for a safer detection apparatus.
Dr. Michel Frenette
Told us to verify if the expression of the oxyS gene, our initial target of choice in E. coli, could be leaky, and advised us to move our target to a plasmid to ensure no leakage of the trigger sequence in the negative control. He also recommended using an inducible promoter to better control trigger expression. While we chose to move away from oxyS, we did move our trigger sequence to a plasmid with an inducible promoter to ensure good control of trigger expression.
Dr. Claude Lemieux and Dre. Monique Turmel
Proposed that we use an infection rather than a transformation to introduce the genetic material in our bacteria, he also said that it would be possible to do so using a lysogenic bacteriophage. He also thought that we could expand our range of detection by using riboregulators activated by metabolites. However, neither of those comments were compatible with the kind of tool we wished to design and use.
Dr. Patrick Lagüe
Said that he could collaborate with our team for the software optimization process and the molecular dynamics. He gave us access to his server for calculation and to host our web tool. He also gave us a lot of great insights into the molecular dynamics simulation, which ended up having a major effect on the design of our project and how we understood ToeHold dynamics.
Dr. Ahmad Saleh
Proposed that we use purified, fragmented chromosomes so that our interrupters may have access to the genetic material more easily. He also told us to consider the possibility that we may have to reduce our chromosomic genetic material to facilitate contact between the genetic material and our switches. He thought that it may be possible to use a thermal shock to liberate the viral genetic material without needing a complex purification step, this idea was also confirmed by Caroline Duchaine, but also pointed out that a high-heat extraction step could affect genetic material integrity.